Risk factors associated with occurrence of placental malaria in a population of parturients in Abeokuta, Ogun State, Nigeria

Background Placental malaria has long been acknowledged as a complication of malaria in pregnancy, and has been associated with poor pregnancy outcome in malaria-endemic areas. This study was conducted to determine the risk factors associated with occurrence of placental malaria in a population of parturients in Abeokuta Ogun State, Nigeria. Materials and Methods Maternal and placenta blood samples were collected from 211 parturients. Blood films were prepared, stained with 10% Giemsa and microscopically analysed for the presence of parasites. Demographic characteristics were recorded in case record forms. Chi-square tests and a regression model were computed to analyse risks, using SPSS version 16.0. Results Overall, 40.8% (86 of 211) of the parturients had malaria at the time of delivery, with 19.0% (40 of 211) having placental malaria. We identified being within the age range of 18-22 years [OR = 4.4, 95% CL = 1.1-17.4, P = 0.046], being primigravid [OR = 2.1, 95% CL = 0.9-5.1, P = 0.028] and living in a congested apartment [OR = 1.6, 95% CL = 0.4-6.0, P = 0.029] as significant risk factors for placental malaria. Non-usage of intermittent preventive treatment (IPT) [OR = 2.6, 95% CL = 1.2-5.4, P = 0.018], long-lasting insecticidal nets (LLINs) [OR = 2.7, 95% CL = 1.3-5.5, P = 0.005] were also risk factors for placental malaria. Conclusions In Abeokuta, the proper use of LLIN and IPT for pregnant women is essential to curb the scourge of malaria, associated risks and poor pregnancy outcomes.


Introduction
Malaria continues to remain one of the most severe and complex health challenges facing the vast majority of tropical and sub-tropical countries around the world.The population groups most at risk of malaria include pregnant women and children under the age of five [1].During pregnancy, the acquired anti-malarial immunity of a woman residing in a malaria-endemic area is affected due to up regulation of the VAR2CSA gene in local Plasmodium falciparum [2].The risk of malaria increases threefold during the second and third trimesters, and fourfold during the initial two post-partum months [3].This increased risk of malaria is related to an alteration in the balance of Th1and Th2-related immune factors [4].
In clinical practice, maternal peripheral parasitaemia is used to detect malaria during pregnancy.However, whereas peripheral parasitaemia may remain below the levels of microscopic detection, these parasites are detectable in the placenta [5,6].Although the mechanism for placental parasitisation is not yet fully known, certain parasite strains have been increasingly detected in pregnant women.It has been suggested that these strains are able to selectively infect the placenta due to their ability to adhere to the chondroitin sulphate A (CS-A) receptor on the syncytiotrophoblast [5,6].
Placental malaria has long been recognised as a complication of malaria in pregnancy in areas of stable trans-mission, and is particularly frequent and more severe in primigravidae [7].Placental malaria is a serious issue in sub-Saharan Africa and is associated with poor pregnancy outcomes, such as low birth weight (LBW), stillbirth and preterm delivery.
Studies have shown that Plasmodium parasites can cross the placenta, leading to mother-to-child transmission of malaria in a condition known as congenital malaria [8,9].Moreover, during delivery, blood vessels may rupture, leading to a mixing of maternal and foetal blood, and transmission of parasites from mothers to children [10].Studies in Africa have shown that at least 7-10% of newborns have malaria parasite-infected placentas, and a significant part of the transmission of parasites from the mother to the child occurs well before the time of delivery [11,12].It is also estimated that 6% of all infant deaths in malaria-endemic areas are a result of malaria infection that took place during the child's prenatal life [13].As pitiful as this situation is, the exact mechanisms for placenta parasitisation and transplacental transmission of malaria parasites are not well understood [5].Therefore, it is very important to identify the risk factors for placental malaria so that pregnant women can make lifestyle choices to reduce the chance of developing malaria.Therefore, this study was conducted to determine risk factors associated with the occurrence of placental malaria in a population of parturients in Abeokuta Ogun State, Nigeria.

Criteria for selection and study population
Only pregnant women in labour (parturients) were included in this study.A total of 211 parturients were randomly selected from the two hospitals, using a formula according to Yamane [14].The sample population was selected irrespective of age, educational background, marital status, occupation, and cultural or religious affiliations.

Ethical clearance/informed consent
The Research and Ethics Committee of the State Hospital Sokenu Ijaiye provided ethical clearance for the study.All medical personnel of the selected hospitals and the participants were fully briefed on the objectives of the study.
Only participants who gave their consent were enrolled in the study.

Collection of placenta blood samples and questionnaire administration
Maternal blood and placenta blood were collected as described by [15].Relevant maternal demographic and clinical characteristics were recorded with the aid of a questionnaire.Information obtained included: age, gravidity, education, and use of intermittent preventive treatment (IPT) and/or long-lasting insecticidal nets (LLINs).

Laboratory procedures
Thick and thin smears were prepared and stained with 10% Giemsa as described by Cheesbrough [16].The blood films were examined microscopically using 100× objectives with oil immersion.Malaria diagnosis was based on identification of asexual stages of Plasmodium spp. on the thick blood film, while thin smears were used for species identification.Slides were declared negative after observing at least 100 high-power fields without detecting any parasites.Parasite density was calculated by estimating parasite numbers/μl of blood from the thick film as described by Greenwood & Armstrong [17].Parasite densities were grouped as low (≤500 parasites/µl), moderate (501-5000 parasites/µl) or high (>5000 parasites/µl).

Statistical analysis
Chi-square tests and logistic regression were used to analyse data, and a probability value of P<0.05 was regarded as significant, using SPSS version 16.0.

Malaria prevalence
Overall, 40.8% (86 of 211) of the parturients had malaria at the time of delivery, with 19.0% (40 of 211) having malaria parasites in their placental blood (placental malaria), with mean maternal peripheral and placenta blood malaria parasite densities of 2597±274 and 3922±561 para- site/µl of blood, respectively.The prevalence of placental malaria decreased with age and gravidity (Table 2).More than 30% of the parturients within the age range of 18-22 years were positive for placenta parasitaemia.The highest prevalence of placental malaria infection (28.4%) was detected among the primigravidae.Placental malaria parasite densities ranged from 26 parasites/µl of blood (i.e.low) to 15375 parasites/µl of blood (high), with the highest proportion showing moderate infection (501 -5000) parasite/µl of blood (Figure 1).
Non-usage of intermittent preventive treatment (IPT) was found to be a significant risk factor for placental malaria [OR = 2.6, 95% CL = 1.2-5.4,P = 0.018], since a higher proportion of the parturients (34.0%) who did not use IPT during pregnancy tested positive for placental malaria infections (Table 3).We also identified non-usage of LLINs as a significant risk factor for placental malaria [OR = 2.7, 95% CL = 1.3-5.5,P = 0.005].Sharing a room with more than one person during pregnancy was also identified as a significant risk factor (P = 0.043) [OR = 2.2, 95% CL = 1.0-4.6],since a higher proportion of the parturients (29.2%) who claimed to have a shared room with more than one person tested positive for placental malaria (Table 3).
The presence of malaria parasite infections in maternal peripheral blood at delivery as a significant risk factor (P = 0.002) for placental malaria [OR = 3.0, 95% CL = 1.5-   2. Demographic characteristics of the parturients associated with risk of placental malaria.

6.1].
There was no significant difference among the parturients that suffered from one or more episodes of malaria infections during pregnancy and those that did not (P>0.05).However, the univariate analysis revealed that the risk of placental malaria infections among this category of parturients was twice as high as in those who did not suffer any malaria attack during pregnancy [OR = 1.9, 95% CL = 0.9-3.8,P = 0.080].Finally, the non-usage of window-and door screening was not a significant risk factor (P = 0.139).All parturients who tested positive for placental malaria claimed to be residing in screened housing even before pregnancy.

Health-seeking behaviour and occurrence of placental malaria
The majority of parturients exercised good health-seeking behaviours by visiting the hospital when they were sick during pregnancy, while the rest used either herbs or drugs prescribed by vendors (poor health-seeking behaviour).All parturients that treated malaria episodes during pregnancy with anti-malaria injections tested positive for both maternal and placental malaria infections (Table 4).Furthermore, the highest prevalence of placental malaria parasite infections was recorded among parturients who treated malaria infections (during pregnancy) with injections, with a prevalence of 3 (100.0%).This was followed by respondents that claimed they did not remember the drugs they used, as they recorded a prevalence of 75% for both maternal and placental malaria infections (Table 4).It is noteworthy, as stated by the respondents, that the antimalaria injections were prescribed by attendants in 'chemist's shops'.Those who did not remember the drugs they used claimed that the drugs were prescribed to them from 'chemist's shops' (Table 4).

Challenges associated with IPT usage
Almost half of the parturients (49.0%) claimed not to use IPT, due to fear of side effects, whereas some complained about the unavailability of the drug during their first and second antenatal visits.Furthermore, some of the respondents claimed not to attach any importance to IPT usage.

Discussion
The epidemiology of placental malaria in Abeokuta Ogun state, Nigeria is similar to that in other parts of Nigeria [6,15].In the current study, women within the age group of 18-22 years as well as primigravidae were more likely to have placental malaria.While the risk of placenta infections is twice as high in primigravidae compared to other gravidities, placental malaria seems to increase fourfold among parturients between the age range of 18-22 years compared with other age groups.This could be attributed to lower levels of acquired immunity among the young women as well as lack of pregnancy-associated and naturally acquired immunity against the placenta-binding parasites in primigravids.Studies have shown that immunity against the placentabinding parasites develops with subsequent pregnancies as a result of previous exposure to the placenta binding strain [18,19].
Living in a single-room apartment as well as the sharing of a room with more than one person during pregnancy were found to be significant risk factors for placental ma- * OR = Odds ratio; CL = Confidence limits laria.This could be related to overcrowding and the frequency of times household doors are opened, which in turn increases the number of mosquitoes that could gain access to the house [20].Studies have also shown that malaria infection can increase with overcrowding, as mosquitoes are attracted to higher levels of carbon dioxide and other body odours present in crowded houses [21].Clinical malaria during pregnancy and maternal malaria parasite infection at parturition increased the risk for placental malaria, with a twofold (non-significant) increase in risk for the former and threefold increase in risk for the latter.In a study carried out in eastern Nigeria, Ezebialu et al. [15] reported similar findings.Pregnant women who had taken precautions and preventive measures during pregnancy had reduced placenta parasitisation, thereby minimizing the effect of malaria parasite infections during pregnancy.
The risk of placental malaria parasite infection in the current study was threefold higher among the parturients that did not use IPT and LLIN, respectively, during pregnancy.Several studies had reported similar findings [18,22,23].However, it is saddening to note that despite the effectiveness of IPT and its administration as directly observed therapy (DOT), most of the parturients that failed to use IPT during pregnancy claimed that they were given IPT, but only pretended to swallow it during their antenatal visits.This situation justifies the need to stress the importance of IPT and LLIN as preventive tools during pregnancy in order to improve compliance.
This study also suggests that the use of window-and door screening might not be very effective in the prevention of malaria infection, due to a number of reasons as stated by Idowu et al. [24], who discovered that the majority of the screens in their study were torn by children or during the process of passing a wire for external antennas, thereby facilitating entry of mosquitoes.
The health-seeking behaviour of women with regards to malaria infection during pregnancy also has an association with susceptibility to placental malaria, as pregnant women that seek treatment with unqualified and untrained 'chemist's shops' recorded the highest prevalence of placental malaria infection.Studies have shown that most of the anti-malarial drugs acquired from chemist shops are of sub-standard quality [25] that have little or no effect against malaria.This practice can significantly increase the risk of placental malaria.

Conclusions
In Abeokuta, approximately one in every five parturients had placental malaria at delivery, with 55% having parasite densities between 501 and 5000 parasites/µl of blood.Women within the age range of 18-22 years and primigravidae were at the highest risk of placental malaria.Other factors that contribute to the risk of placenta infections included non-usage of LLIN and IPT, poor housing conditions and poor health-seeking behaviour.

Figure 1 .
Figure 1.Parasite density in the placenta of 40 parturients with placental malaria.

Table 4 .
Health seeking behaviour and occurrence of maternal and placental malaria infections in 70 parturients.* Treatment administered by chemist attendants or drug vendors, rather than doctors

Table 1 .
Characteristics of the respondents

Table 3 .
Risk factors for placental malaria among parturients.